Macromolecular x-ray crystallography is an essential aspect of modern drug discovery and molecular biology. Using x-ray crystallographic techniques, the three-dimensional structures of biological macromolecules, such as proteins, nucleic acids, and their various complexes, can be determined at practically atomic level resolution. The enormous value of three-dimensional information has led to a growing demand for innovative products in the area of protein crystallization, which is currently the major rate limiting step in x-ray structure determination.
One of the first and most important steps of the x-ray crystal structure determination of a target macromolecule is to grow large, well diffracting crystals with the macromolecule. As techniques for collecting and analyzing x-ray diffraction data have become more rapid and automated, crystal growth has become a rate limiting step in the structure determination process.
Vapor diffusion is the most widely used technique for crystallization in modern macromolecular x-ray crystallography. In this technique, a small volume of the macromolecule sample is mixed with an approximately equal volume of a crystallization solution. The resulting drop of liquid (containing macromolecule and dilute crystallization solution) is sealed in a chamber with a much larger reservoir volume of the crystallization solution. The drop is kept separate from the reservoir, either by hanging from a glass cover slip or by sitting on a tiny pedestal. Over time, the crystallization drop and the reservoir solutions equilibrate via vapor diffusion of the volatile species. Supersaturating concentrations of the macromolecule are achieved, resulting in crystallization in the drop when the appropriate reservoir solution is used.
The process of growing biological macromolecule crystals remains, however, a highly empirical process. Macromolecular crystallization is a hyperdimensional phenomena, dependent on a host of experimental parameters including pH, temperature, and the concentration of salts, macromolecules, and the particular precipitating agent (of which there are hundreds). A sampling of this hyperspace, via thousands of crystallization trials, eventually leads to the precise conditions for crystal growth. Thus, the ability to rapidly and easily generate many crystallization trials is important in determining the right conditions for crystallization. Also, since so many multidimensional data points are generated in these crystallization trials, it is imperative that the experimenter be able to accurately record and analyze the data so that promising conditions are pursued, while no further time, resources, and effort are spent on negative conditions.
Recently, an international protein structure initiative has taken shape with the goal of determining the three dimensional structures of all representative protein folds. This massive undertaking in structural biology which may some day rival the human genome sequencing project in size and scope, is estimated to require a minimum of 100,000 x-ray structure determinations of newly discovered proteins for which no structural information is currently available or predicted. For perspective, the total number of reported novel crystal structures determined to date (spanning nearly 50 years of work) is only approximately 10,000.
Using existing methods for the crystallization of proteins (random screens of conditions), the protein structure initiative will require a minimum of approximately 100 million crystallization trials. In addition, the biological information gleaned from genomic research in the protein structure initiative are expected to create even more demand for structural information. Specifically, the biotechnology and pharmaceutical industries are estimated to require upwards of ten fold more protein crystallization experiments (one billion) as a result of research and structure based drug design and the use of crystallized therapeutic proteins. This would require that each of the approximately 500 macromolecular crystallography labs worldwide be responsible for setting up approximately 2000 crystallization trials every working day of the year for five years. Currently, there is no known device available for setting up analysis macromolecular crystallization data on this scale.
The preparation of crystal growth screening solutions is a tedious and time consuming endeavor. As such, high-throughput crystal growth demands that the construction of crystallization screening solutions be fully automated. To address this issue, the inventors have developed a method and system, an embodiment of which is called a xe2x80x9cMatrix Makerxe2x80x9d, for creating new crystallization screening solutions in a crystallization plate (drawing from, for example, 96 different stock solutions). A variation of the invention (xe2x80x9cDrop Makerxe2x80x9d) is capable of setting up crystallization drops in the plate once the screening solutions have been prepared in the plate.
Another embodiment of the invention is capable of running chromatographic protein purification experiments by aspirating crude cell extracts from a sample plate and pumping them over a plurality of chromatography devices such as chromatography cartridges or columns. The chromatography devices are then washed by pumping a plurality of different elution buffers over the chromatography devices and collecting the liquids that flow through the chromatography devices into recipient containers. a single valve port serving as both inlet port and outlet port, and the connected pin being both a dispensing pin and an aspiration pin.
According to an embodiment of the present invention, a system for mixing crystallization trial matrices includes a plurality of precision pumps (such as precision syringe pumps), a distributor and a processor system, which may contain one or more computer or digital processors. Each pump draws, under the control of the processor, an associated stock solution from a stock solution source, and pumps the drawn stock solution out through an outlet. The distributor, also under the control of the processor system, directs a stock solution from a particular pump outlet to a selected solution receptacle or holder.
A multi-port distribution valve may be associated with each precision pump. Each valve, under control of the processor system, can at any time connect its associated pump to one of the inlets or outlets.
In one embodiment, individual inlets of a particular pump may be connected to different stock solutions. Each outlet of a pump may be uniquely associated with an inlet, such that a particular stock solution always enters through one of said inlets and always exits through the associated outlet. Furthermore, each pump may have an inlet connected to a water/wash source, and an outlet for disposing of waste.
In one embodiment, the distributor comprises one or more outlet manifolds which hold an array of dispensing pins that are connected to the outlet ports, and positioning means for aligning a particular pin over the desired solution receptacle. The dispensing pins may be made of stainless steel or some other suitable material. The distributor may also have an array of pins that are connected to tubing that is connected to one of the pump inlets. The pins and their associated lines may be used to aspirate or dispense liquids from solution receptacle container plates located beneath the distributor.
The positioning means may include a gantry on which the outlet manifold is supported. The processor system may control the movement of the gantry in two or three dimensions. In one embodiment, multiple gantries may be used.
Solution receptacles may be test tubes, crystallization plate wells, or other suitable containers (for example, Society for Biomolecular Screening type plasticware devices) that may be, for example, arranged in an array.
In one embodiment, the processor controls the pumps, valves and gantry according to predefined recipes that describe which solutions are to be mixed, each destination solution receptacle, and solution volumes. These recipes may be viewed and edited by a user.
In another embodiment, the processor may control the pumps, valves and gantry according to predefined protocols for purifying proteins chromatographically or for setting up crystallization plates. The protocols may be viewed and edited by a user.